Simian virus 40 DNA replication in vitro.

TLDR

The assay enables measurement of SV40 T antigen activity and supports purification of cellular proteins involved in DNA replication. Soluble extracts from infected or uninfected monkey cells drive origin‑dependent plasmid replication, forming theta intermediates and covalently closed circular DNA, with activity requiring T antigen in uninfected extracts and being sensitive to aphidicolin. The system replicates plasmids containing the SV40 origin efficiently, requires T antigen for uninfected extracts, is abolished by a 4‑bp deletion, produces theta structures, is aphidicolin‑sensitive, ddTTP‑resistant, and yields covalently closed circular DNA, closely mirroring in vivo SV40 replication.

Abstract

Soluble extracts prepared from monkey cells (COS-1 or BSC-40) infected with simian virus 40 (SV40) catalyze the efficient replication of exogenously added plasmid DNA molecules containing the cloned SV40 origin of replication. Extracts prepared from uninfected monkey cells also support origin-dependent replication in vitro but only in the presence of added SV40 large tumor (T) antigen. Very little DNA synthesis is observed when the cloned viral origin contains a 4-base-pair deletion mutation known to abolish SV40 DNA replication in vivo or when the parental plasmid vector lacking SV40 sequences is employed as template. The in vitro replication reaction proceeds via branched intermediates (theta structures) that resemble in vivo replication intermediates. Replication is sensitive to aphidicolin but relatively resistant to dideoxythymidine triphosphate. The product of the reaction consists of covalently closed circular DNA molecules that contain full-length daughter strands hydrogen bonded to the parental template. These observations support the conclusion that replication in the in vitro system closely resembles SV40 DNA replication in vivo. The system provides a biochemical assay for the replication activity of SV40 T antigen and should also facilitate the purification and functional characterization of cellular proteins involved in DNA replication.